Stereochemical requirements for receptor recognition of the mu-opioid peptide endomorphin-1

A series of diastereoisomers of endomorphin-1 (EMI, Tyr(1)-Pro(2)-Trp(3)-Ph e(4)-NH2) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe(4)]EM1 possessed 1/10 the potency of EM1, while po tencies of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 were 50- and 100-fold lower, res pectively. Drastic loss of activity occurred in the [D-Pro(2)]EM1 peptide. The structural determinants for the inactivity and reduced potency of the d iastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro(2)]EM1 using NOE-derived distance co nstraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D-Pro(2)]EM1 was explai ned by structural comparison with EMI (Podlogar et al.,1998, FEES Lett 439: 13-20). The two peptides showed an opposite orientation of the Trp(3) resid ue with respect to Tyr(1), thus suggesting a role of Pro(2) as a stereochem ical spacer in orienting Trp(3) and Phe(4) toward regions suitable for mu-r eceptor interaction. The agonist activity of [D-Tyr(1)]EM1 and [D-Trp(3)]EM 1 was attributed to their ability to adopt low-energy conformations that m imic those of EM 1. The requirements for mu-receptor activation were examin ed further by comparing EMI with the mu-peptide [D-Ala(2), MePhe(4), Gly-ol ]-enkephalin (DAMGO). Conformations of DAMGO with a Tyr(1)-MePhe(4) phenyl ring separation of similar to 12 Angstrom were found to mimic Tyr(1)-Phe(4) of EMI, thus suggesting overlapping binding modes between these two peptid es.